(1) Field of the Invention
The present invention generally relates to methods and compositions for increasing enzyme activities. More particularly, the invention provides methods and compositions useful for increasing SIR2 deacetylation activity.
(2) Description of the Related Art
References Cited
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The SIR2 (Silent Information Regulator) enzymes (also known as sirtuins) make up a newly classified family of NAD+-dependent protein deacetylases that employ metabolically valuable NAD+ as a substrate to convert acetyllysine sidechains to unmodified lysine sidechains in protein co-substrates (Landry et al., 2000a; Imai et al., 2000). The yeast SIR2 proteins were originally identified as co-regulators of genetic silencing and are localized at chromatin in protein modules called SIR complexes. Within SIR complexes these enzymes are believed to regulate chromatin structure (Smith et al., 2000; Rine & Herskowitz, 1987) by establishment and maintenance of hypoacetylation at H3 and H4 histone N-terminal tails (Rusche et al., 2003; Anderson et al., 2003; Braunstein et al., 1993). The role of these enzymes in regulating genetic information as part of a potent DNA-repressing machinery emphasizes their importance to the cell. Indeed, the SIR2 enzymes are broadly distributed across all phyla of life (Brachmann et al., 1995; Smith et al., 2000) and appear to have roles in the regulation of lifespan (Lin et al., 2000; Tissenbaum & Guarente, 2000) and genomic stability (Brachmann et al., 1995). For example, SIR2 has been identified as essential to life-span extension caused by calorie restriction in S. cerevisiae (Lin et al., 2000), C. elegans (Tissenbaum & Guarente, 2000) and impacts lifespan in Drosophila (Astrom et al., 2003). Lifespan extension is caused by an increase of SIR2 activity during calorie restriction since additional copies of SIR2 genes confer an increased longevity phenotype in S. cerevisiae (Lin et al., 2000) and in C. elegans (Tissenbaum & Guarente, 2000). Since calorie restriction also confers benefits associated with increased lifespan in mammals, including primates (Lin et al., 2000), increased SIR2 activity likely leads to increased longevity in mammals.
The mechanism by which SIR2 is activated by caloric restriction is not well understood, but increased NAD+/NADH ratio or increased NAD+ concentration have been suggested (Lin & Guarente, 2002; Campisi, 2000). A role for nicotinamide and the gene PNC1 in regulating SIR2 activity has also been demonstrated (Anderson et al., 2003; Bitterman et al., 2002; Anderson et al., 2002). PNC1 deamidates nicotinamide to form nicotinic acid and can lower levels of nicotinamide formed as a product of SIR2 and in pathways of NAD+metabolism (Anderson et al., 2003; Bitterman et al., 2002; Anderson et al., 2002). PNC1 is overexpressed in several stress conditions (Anderson et al., 2002; Sinclair, 2002) that increase longevity in yeast, implying that increased PNC1 activity increases SIR2 action by reducing nicotinamide inhibition. Nicotinamide is a potent inhibitor of SIR2 enzyme activity (Bitterman et al., 2002; Landry et al., 2000b) and also serves as a base-exchange substrate of SIR2 enzymes (Landry et al., 2000b; Min et al., 2001; Sauve et al., 2001). The relationship between nicotinamide base-exchange, nicotinamide inhibition and the reaction mechanism of SIR2 has not been defined, but is fundamental to regulation of SIR2 in vivo.
Further characterization of the SIR2 reaction mechanism is needed to help determine ways that the deacetylation reaction could be enhanced. The present invention satisfies that need, and identifies various compounds that promote the deacetylation reaction in the presence of otherwise inhibiting amounts of nicotinamide.